The native structure of the IgG immunoglobulin class consists of a covalently linked homodimer comprised of two heavy and two light chains. Thus a monoclonal IgG will bind two molecules of antigen simultaneously (monospecific bivalency). It is preferred for some therapeutic purposes that the IgG bind only a single antigen molecule (monovalency) or bind two different antigens simultaneously (bispecific bivalency) while retaining the long half-life and potential for effector function of the native IgG immunoglobulin. For these purposes the molecular properties of the IgG need to be engineered to prevent homodimerization. Methods described in the art for preventing homodimerization have either not been adequately efficient or give less stable products (Merchant et al 1998 Nature Biotechnology 16:677; Gunasekaran et al 2010 J. Biol. Chem. 285:19637; Muda et al 2011 Proten Engineering, Design, and Selection 24:447).
It is critical in designing a new method for achieving heterodimerization versus homodimerization that the desirable attributes of the Fc domain of the IgG be retained. The Fc region of an immunoglobulin mediates effector functions that have been divided into two categories. In the first are functions that occur independently of antigen binding; these functions confer persistence in circulation and the ability to be transferred across cellular barriers by transcytosis (see Ward and Ghetie, Therapeutic Immunology 2:77-94, 1995, Capon et al. Nature 1989). The circulatory half-life of the IgG subclass of immunoglobulins is regulated by the affinity of the Fc region for the neonatal Fc receptor or FcRn (see Ghetie et al., Nature Biotechnol. 15:637-640, 1997; Kim et. al., Eur. J. Immunol. 24:542-548, 1994; Dall'Acqua et al. (J. Immunol. 169:5171-5180, 2002). The second general category of effector functions include those that operate after an immunoglobulin binds an antigen. In the case of IgG, these functions involve the participation of the complement cascade or Fc gamma receptor (FcγR)-bearing cells. Binding of the Fc region to an FcγR causes certain immune effects, for example, endocytosis of immune complexes, engulfment and destruction of immunoglobulin-coated particles or microorganisms (also called antibody-dependent phagocytosis, or ADCP), clearance of immune complexes, lysis of immunoglobulin-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, regulation of immune system cell activation, and regulation of immunoglobulin production.
Certain engineered binding polypeptides (e.g., antibody variants (e.g., scFvs) or antibody fragments (e.g., Fab fragments)), while benefiting from their smaller molecular size and/or monovalency, also suffer several disadvantages attributable to the absence of a functional Fc region. For example, Fab fragments have short half-lives in vivo because they lack the Fc region that is required for FcRn binding and are rapidly filtered out of the blood by the kidneys owing to their small size.
Accordingly, there is a need for Fc-containing binding molecules which can be produced and isolated efficiently and robustly while retaining desired Fc effector function(s) and providing desired valencies and specificities.